For the car sequencing（CS） problem, the draw-backs of the ＂sliding windows＂ technique used in the objective function have not been rectified, and no high quality initial solution has been acquired to accelerate th...For the car sequencing（CS） problem, the draw-backs of the ＂sliding windows＂ technique used in the objective function have not been rectified, and no high quality initial solution has been acquired to accelerate the improvement of the solution quality. Firstly, the objective function is improved to solve the double and bias counting of violations broadly discussed. Then, a new method combining heuristic with constraint propagation is proposed which constructs initial solutions under a parallel framework. Based on constraint propagation, three filtering rules are designed to intersecting with three greedy functions, so the variable domain is narrowed in the process of the construction. The parallel framework is served to show its robustness in terms of the quality of the solution since it greatly increases the performance of obtaining the best solution. In the computational experiments, 109 instances of 3 sets from the CSPLib＇ s benchmarks are used to test the performance of the proposed method. Experiment results show that the proposed method outperforms others in acquiring the best-known results for 85 best-known results of 109 are obtained with only one construction. The proposed research provides an avenue to remedy the deficiencies of ＂sliding windows＂ technique and construct high quality initial solutions.展开更多
The microbiome has a profound impact on host fitness. pH, oxygen, nutrients, or other factors such as food or pharmaceuticals, subject the microbiome to variations in the gastrointestinal tract. This variation is a ca...The microbiome has a profound impact on host fitness. pH, oxygen, nutrients, or other factors such as food or pharmaceuticals, subject the microbiome to variations in the gastrointestinal tract. This variation is a cause for concern given dysbiosis of the microbiome is correlated with various disease states. Currently, much research relies on model organisms to study microbial communities since intact microbiomes are challenging to utilize. The objective of this study is to culture an explanted colon microbiome of 4 Balb/c mice to develop an in vitro tool for future microbiome studies. We cultured homogenates of the distal colons of 4 mice in trans-well culture dishes. These dishes were incubated for 24 hours in two different oxygen concentration levels and the pH was compared before and after incubation of the cultures. To analyze the integrity of the microbiome, we utilized massively paralleled DNA sequencing with 16S metagenomics to characterize fecal and colon samples to speculate whether future studies may utilize feces in constructing an in vitro microbial community to spare animal lives. We found that pH and familial relationships had a profound impact on community structure while oxygen did not have a significant influence. The feces and the colon were similar in community profiles, which lends credence to utilizing feces in future studies. The gut microbiome is of great interest and great importance for studies in a variety of different diseases. Many laboratories do not have access to germ-free mice, which is one optimal way to study mammalian microbiomes, but this technique allowed for the in vitro culturing of a majority of the prokaryotes isolated from the colons of mice. This may allow an alternative to study the interactions of this very diverse population of microorganisms without the need for germ-free conditions.展开更多
Massively parallel sequencing （MPS） technology is capable of determining the sizes of short tandem repeat （STR） alleles as well as their individual nueleotide sequences. Thus, single nucleotide polymorphisms （SNP...Massively parallel sequencing （MPS） technology is capable of determining the sizes of short tandem repeat （STR） alleles as well as their individual nueleotide sequences. Thus, single nucleotide polymorphisms （SNPs） within the repeat regions of STRs and variations in the pattern of repeat units in a given repeat motif can be used to differentiate alleles of the same length. In this study, MPS was used to sequence 28 forensically-relevant Y-chromosome STRs in a set of 41 DNA samples from the 3 major U.S. population groups （African Americans, Caucasians, and Hispanics）. The resulting sequence data, which were analyzed with STRait Razor v2.0, revealed 37 unique allele sequence variants that have not been previously reported. Of these, 19 sequences were variations of documented sequences resulting from the presence of intra-repeat SNPs or alternative repeat unit patterns. Despite a limited sampling, two of the most frequently-observed variants were found only in African American samples. The remaining 18 variants represented allele sequences for which there were no published data with which to compare. These findings illustrate the great potential of MPS with regard to increasing the resolving power of STR typing and emphasize the need for sample population characterization of STR alleles.展开更多
Genetic profiling is a standard procedure for human identification,i.e.in criminal cases and mass disasters,and has been proven to be an important part in the process in the repatriation of victims to their relatives....Genetic profiling is a standard procedure for human identification,i.e.in criminal cases and mass disasters,and has been proven to be an important part in the process in the repatriation of victims to their relatives.In the event of a catastrophe whether it be a natural disaster,terror attack or accident,fatalities of many nationalities may be a consequence and international collaboration becomes necessary.Current DNA techniques used on a routine basis at forensic laboratories world-wide are very useful,and results reported from different labs are compared,making it possible to be matched in order to declare the identification of a victim.Statistical calculations of possibilities of a random match are achievable since population data from many parts of the world are available.However,decomposition and degradation of the remains are not uncommon in the aftermath of a catastrophe and hence it may be difficult to retrieve detailed DNA profiles from such samples.Massive parallel sequencing(MPS)is a technique capable of producing a vast amount of DNA sequence data in a high-through put manner,and panels of single nucleotide polymorphism(SNP)markers allow the amplification of small DNA fragments,often seen in compromised samples.Here,we report the results from a set of 10 samples from missing person identification cases,analyzed with an MPS based method comprising 131 SNP markers and compared with direct reference material or buccal swab samples collected from relatives of the deceased.We assess the weight of evidence of a match by statistical calculation.Furthermore,we compare results reported on different platforms using different SNP panels,and conclude that more work has to be done if results from missing person identification cases analyzed on MPS with SNP panels at different laboratories are to be fully reliable and thus comparable.展开更多
The custom-designed single nucleotide polymorphism(SNP)panel amplified 231 autosomal SNPs in one PCR reaction and subsequently sequenced with massively parallel sequencing(MPS)technology and Ion Torrent personal genom...The custom-designed single nucleotide polymorphism(SNP)panel amplified 231 autosomal SNPs in one PCR reaction and subsequently sequenced with massively parallel sequencing(MPS)technology and Ion Torrent personal genome machine(PGM).SNPs were chosen from SNPforID,IISNP,HapMap,dbSNP,and related published literatures.Full concordance was obtained between available MPS calling and Sanger sequencing with 9947A and 9948 controls.Ten SNPs(rs4606077,rs334355,rs430046,rs2920816,rs4530059,rs1478829,rs1498553,rs7141285,rs12714757 and rs2189011)with low coverage or heterozygote imbalance should be optimized or excluded from the panel.Sequence data had sufficiently high coverage and gave reliable SNP calling for the remaining 221 loci with the custom MPS-SNP panel.A default DNA input amount of 10 ng per reaction was recommended by Ampliseq technology but sensitivity testing revealed positive results from as little as 1 ng input DNA.Mixture testing with this panel is possible through analysis of the F MAR(frequency of major allele reads)values at most loci with enough high coverage depth and low level of sequencing noise.These results indicate the potential advantage of the custom MPS-SNP assays and Ion Torrent PGM platform for forensic study.展开更多
The field of forensic DNA typing,often referred to as“DNA fingerprinting,”has evolved and expanded considerably since its beginnings in the mid-1980s.Originally,forensic DNA typing was primarily used for individual ...The field of forensic DNA typing,often referred to as“DNA fingerprinting,”has evolved and expanded considerably since its beginnings in the mid-1980s.Originally,forensic DNA typing was primarily used for individual identification and criminal investigations,but it has evolved into a versatile discipline with a wide range of applications.This article addresses the growing scope of forensic genetics,which includes advances in DNA sequencing technologies,mixture analysis,body fluid identification,phenotypic profiling,forensic genealogy,microbiological analysis,exploration of novel markers,and ethical and legal considerations.These developments have enabled the analysis of difficult samples and provided comprehensive insights into the origins of biological evidence.In an ever-evolving landscape,forensic genetics continues to shape the future of forensic science by providing new tools and techniques that help deliver justice in an increasingly complex world.展开更多
Research has shown that the hypomagnetic field(HMF)can affect embryo development,cell proliferation,learning and memory,and in vitro tubulin assembly.In the present study,we aimed to elucidate the molecular mechanism ...Research has shown that the hypomagnetic field(HMF)can affect embryo development,cell proliferation,learning and memory,and in vitro tubulin assembly.In the present study,we aimed to elucidate the molecular mechanism by which the HMF exerts its effect,by comparing the transcriptome profiles of human neuroblastoma cells exposed to either the HMF or the geomagnetic field.A total of 2464 differentially expressed genes(DEGs)were identified,216 of which were up-regulated and2248 of which were down-regulated after exposure to the HMF.These DEGs were found to be significantly clustered into several key processes,namely macromolecule localization,protein transport,RNA processing,and brain function.Seventeen DEGs were verified by real-time quantitative PCR,and the expression levels of nine of these DEGs were measured every 6 h.Most notably,MAPK1 and CRY2,showed significant up-and down-regulation,respectively,during the first 6 h of HMF exposure,which suggests involvement of the MAPK pathway and cryptochrome in the early bio-HMF response.Our results provide insights into the molecular mechanisms underlying the observed biological effects of the HMF.展开更多
Next-generation sequencing （NGS） technologies allow the cost-effective sequencing of whole genomes and have expanded the scope of genomics to novel applications, such as the genome-wide characterization of intraspec...Next-generation sequencing （NGS） technologies allow the cost-effective sequencing of whole genomes and have expanded the scope of genomics to novel applications, such as the genome-wide characterization of intraspecific polymorphisms and the rapid mapping and identification of point mutations. Next-generation sequencing platforms, such as the Illumina HiSeq2ooo platform, are now commercially available at affordable prices and routinely produce an enormous amount of sequence data, but their wide use is often hindered by a lack of knowledge on how to manipulateand process the information produced. In this review, we focus on the strategies that are available to geneticists who wish to incorporate these novel approaches into their research but who are not familiar with the necessary bioinformatic concepts and computational tools. In particular, we comprehensively summarize case studies where the use of NGS technologies has led to the identification of point mutations, a strategy that has been dubbed ＂mapping-by-sequencing＇, and review examples from plants and other model species such as Caenorhabditis elegans, Saccharomyces cerevisiae, and Drosophila mela- nogaster. As these technologies are becoming cheaper and more powerful, their use is also expanding to allow mutation identification in species with larger genomes, such as many crop plants.展开更多
With the development and maturation of massively parallel sequencing(MPS)technology,the mitochondrial genome(mitogenome)sequencing is increasingly applied in the forensic field.In this study,we employed the strategy o...With the development and maturation of massively parallel sequencing(MPS)technology,the mitochondrial genome(mitogenome)sequencing is increasingly applied in the forensic field.In this study,we employed the strategy of short overlapping amplicons for the whole mitogenome,library preparation with tagmentation using the Nextera®XT DNA Library Preparation Kit,sequencing on the MiSeq FGxTM Forensic Genomics System and analyzing data using the mitochondrial(mtDNA)MSR Plug-in and the mtDNA Variant Analyzer.A total of 27 libraries and 56 libraries were sequenced in a run using MiSeq Reagent Kit v2 and v3,respectively.Results showed more than 1800×of averaged depth of coverage(DoC)at each position.Concordant haplotypes of 9947 A and 2800 M were obtained at 32 variants.Cross-reactivity was observed with 1 ng primate DNA and 10 ng non-primate DNA but could be easily distinguished.Full and accurate variants were obtained from at least 50 pg input DNA and from minor contributors between 19:1 and 1:19 mixed ratios with known reference profiles.More than 86%variants were detected from≥200-bp degraded samples but its haplotype was assigned to more ancestral haplogroup.Further,a total of 3962 variants were observed at 613 nucleotide positions from 103 Xibe mitogenomes with 25:1 ratio of transitions to transversions.Two new transversions(C13735A and A14755C)and two tri-alleles at nps 9824 and 16092 were identified.There were 103 unique mitogenome haplotypes from 103 Chinese Xibe that were assigned to 79 haplogroups.Haplogroup D was the preponderant top-level haplogroup in Xibe followed by F,B,M,A,N,G,C,Z,Y,HV and J.Random match probability(RMP)and haplotype diversity(HD)of the whole mitogenome was calculated as 0.0097 and 1.0000,respectively.Compared with HVS-I only,RMP decreased 33.56%,while the number of haplotypes and HD increased 15.73%and 0.49%,respectively.Principal component analysis(PCA)showed that Xibe was clustered to East and Southeast Asian.As a whole,this MPS strategy is suitable for the whole mitogenome sequencing especially for degraded samples and can facilitate generating mitogenome data to support the routine application in forensic sciences.EMP00726 is the first whole mitogenome dataset from Xibe contributed to the EMPOP.展开更多
To evaluate the promising advantages of massively parallel sequencing(MPS)in our casework,we analysed a total of 33 Y-chromosomal short tandem repeats(Y-STRs)with traditional capillary electrophoresis(CE)and 25 Y-STRs...To evaluate the promising advantages of massively parallel sequencing(MPS)in our casework,we analysed a total of 33 Y-chromosomal short tandem repeats(Y-STRs)with traditional capillary electrophoresis(CE)and 25 Y-STRs using the newer MPS technology.We studied the outcome of both technologies in 64 father-son pairs using stock and custom-designed kits.Current MPS technology confirmed the 13 mutational events observed with CE and improved our understanding of the complex nature of STR mutations.By detecting isometric sequence variants between unrelated males,we show that sequencing Y-STRs using MPS can boost discrimination power.展开更多
Massively parallel sequencing(MPS)offers a useful alternative to capillary electrophoresis(CE)based analysis of human identification markers in forensic genetics.By sequencing short tandem repeats(STRs)instead of dete...Massively parallel sequencing(MPS)offers a useful alternative to capillary electrophoresis(CE)based analysis of human identification markers in forensic genetics.By sequencing short tandem repeats(STRs)instead of determining the fragment lengths by CE,the sequence variation within the repeat region and the flanking regions may be identified.In this study,we typed 264 Uyghur individuals using the MiSeq FGx^(^(TM)) Forensic Genomics System and Primer Mix A of the ForenSeq^(^(TM)) DNA Signature Prep Kit that amplifies 27 autosomal STRs,25 Y-STRs,seven X-STRs,and 94 HID-SNPs.STRinNGS v.1.0 and GATK 3.6 were used to analyse the STR regions and HID-SNPs,respectively.Increased allelic diversity was observed for 33 STRs with the PCR-MPS assay.The largest increases were found in DYS389II and D12S391,where the numbers of sequenced alleles were 3–4 times larger than those of alleles determined by repeat length alone.A relatively large number of flanking region variants(28 SNPs and three InDels)were observed in the Uyghur population.Seventeen of the flanking region SNPs were rare,and 12 of these SNPs had no accession number in dbSNP.The combined mean match probability and typical paternity index based on 26 sequenced autosomal STRs were 3.85E36 and 1.49Eþ16,respectively.This was 10000 times lower and 1000 times higher,respectively,than the same parameters calculated from STR repeat lengths.展开更多
基金Supported by National Natural Science Foundation of China(Grant Nos.51435009,71302085)Zhejiang Provincial Natural Science Foundation of China(Grant No.LQ14E080002)K.C.Wong Magna Fund in Ningbo University
文摘For the car sequencing（CS） problem, the draw-backs of the ＂sliding windows＂ technique used in the objective function have not been rectified, and no high quality initial solution has been acquired to accelerate the improvement of the solution quality. Firstly, the objective function is improved to solve the double and bias counting of violations broadly discussed. Then, a new method combining heuristic with constraint propagation is proposed which constructs initial solutions under a parallel framework. Based on constraint propagation, three filtering rules are designed to intersecting with three greedy functions, so the variable domain is narrowed in the process of the construction. The parallel framework is served to show its robustness in terms of the quality of the solution since it greatly increases the performance of obtaining the best solution. In the computational experiments, 109 instances of 3 sets from the CSPLib＇ s benchmarks are used to test the performance of the proposed method. Experiment results show that the proposed method outperforms others in acquiring the best-known results for 85 best-known results of 109 are obtained with only one construction. The proposed research provides an avenue to remedy the deficiencies of ＂sliding windows＂ technique and construct high quality initial solutions.
文摘The microbiome has a profound impact on host fitness. pH, oxygen, nutrients, or other factors such as food or pharmaceuticals, subject the microbiome to variations in the gastrointestinal tract. This variation is a cause for concern given dysbiosis of the microbiome is correlated with various disease states. Currently, much research relies on model organisms to study microbial communities since intact microbiomes are challenging to utilize. The objective of this study is to culture an explanted colon microbiome of 4 Balb/c mice to develop an in vitro tool for future microbiome studies. We cultured homogenates of the distal colons of 4 mice in trans-well culture dishes. These dishes were incubated for 24 hours in two different oxygen concentration levels and the pH was compared before and after incubation of the cultures. To analyze the integrity of the microbiome, we utilized massively paralleled DNA sequencing with 16S metagenomics to characterize fecal and colon samples to speculate whether future studies may utilize feces in constructing an in vitro microbial community to spare animal lives. We found that pH and familial relationships had a profound impact on community structure while oxygen did not have a significant influence. The feces and the colon were similar in community profiles, which lends credence to utilizing feces in future studies. The gut microbiome is of great interest and great importance for studies in a variety of different diseases. Many laboratories do not have access to germ-free mice, which is one optimal way to study mammalian microbiomes, but this technique allowed for the in vitro culturing of a majority of the prokaryotes isolated from the colons of mice. This may allow an alternative to study the interactions of this very diverse population of microorganisms without the need for germ-free conditions.
基金supported in part by the grant‘‘Development of Reference Sample DNA Profiling for Databases Using Next Generation Sequencing Technologies"(Award No.2012-DNBXK033)awarded to BB by the National Institute of Justice,Office of Justice Programs,U.S
文摘Massively parallel sequencing （MPS） technology is capable of determining the sizes of short tandem repeat （STR） alleles as well as their individual nueleotide sequences. Thus, single nucleotide polymorphisms （SNPs） within the repeat regions of STRs and variations in the pattern of repeat units in a given repeat motif can be used to differentiate alleles of the same length. In this study, MPS was used to sequence 28 forensically-relevant Y-chromosome STRs in a set of 41 DNA samples from the 3 major U.S. population groups （African Americans, Caucasians, and Hispanics）. The resulting sequence data, which were analyzed with STRait Razor v2.0, revealed 37 unique allele sequence variants that have not been previously reported. Of these, 19 sequences were variations of documented sequences resulting from the presence of intra-repeat SNPs or alternative repeat unit patterns. Despite a limited sampling, two of the most frequently-observed variants were found only in African American samples. The remaining 18 variants represented allele sequences for which there were no published data with which to compare. These findings illustrate the great potential of MPS with regard to increasing the resolving power of STR typing and emphasize the need for sample population characterization of STR alleles.
文摘Genetic profiling is a standard procedure for human identification,i.e.in criminal cases and mass disasters,and has been proven to be an important part in the process in the repatriation of victims to their relatives.In the event of a catastrophe whether it be a natural disaster,terror attack or accident,fatalities of many nationalities may be a consequence and international collaboration becomes necessary.Current DNA techniques used on a routine basis at forensic laboratories world-wide are very useful,and results reported from different labs are compared,making it possible to be matched in order to declare the identification of a victim.Statistical calculations of possibilities of a random match are achievable since population data from many parts of the world are available.However,decomposition and degradation of the remains are not uncommon in the aftermath of a catastrophe and hence it may be difficult to retrieve detailed DNA profiles from such samples.Massive parallel sequencing(MPS)is a technique capable of producing a vast amount of DNA sequence data in a high-through put manner,and panels of single nucleotide polymorphism(SNP)markers allow the amplification of small DNA fragments,often seen in compromised samples.Here,we report the results from a set of 10 samples from missing person identification cases,analyzed with an MPS based method comprising 131 SNP markers and compared with direct reference material or buccal swab samples collected from relatives of the deceased.We assess the weight of evidence of a match by statistical calculation.Furthermore,we compare results reported on different platforms using different SNP panels,and conclude that more work has to be done if results from missing person identification cases analyzed on MPS with SNP panels at different laboratories are to be fully reliable and thus comparable.
基金supported by grants from the National Natu-ral Science Foundation of China[grant number 81330073],[grant number 81302620]the Ministry of Science and Technology of China[grant number 2016YFC0800703]the Science and Technology Commission of Shanghai Municipality[grant number 14DZ2270800].
文摘The custom-designed single nucleotide polymorphism(SNP)panel amplified 231 autosomal SNPs in one PCR reaction and subsequently sequenced with massively parallel sequencing(MPS)technology and Ion Torrent personal genome machine(PGM).SNPs were chosen from SNPforID,IISNP,HapMap,dbSNP,and related published literatures.Full concordance was obtained between available MPS calling and Sanger sequencing with 9947A and 9948 controls.Ten SNPs(rs4606077,rs334355,rs430046,rs2920816,rs4530059,rs1478829,rs1498553,rs7141285,rs12714757 and rs2189011)with low coverage or heterozygote imbalance should be optimized or excluded from the panel.Sequence data had sufficiently high coverage and gave reliable SNP calling for the remaining 221 loci with the custom MPS-SNP panel.A default DNA input amount of 10 ng per reaction was recommended by Ampliseq technology but sensitivity testing revealed positive results from as little as 1 ng input DNA.Mixture testing with this panel is possible through analysis of the F MAR(frequency of major allele reads)values at most loci with enough high coverage depth and low level of sequencing noise.These results indicate the potential advantage of the custom MPS-SNP assays and Ion Torrent PGM platform for forensic study.
基金supported by grants from the National Natural Science Foundation of China(No.82030058).
文摘The field of forensic DNA typing,often referred to as“DNA fingerprinting,”has evolved and expanded considerably since its beginnings in the mid-1980s.Originally,forensic DNA typing was primarily used for individual identification and criminal investigations,but it has evolved into a versatile discipline with a wide range of applications.This article addresses the growing scope of forensic genetics,which includes advances in DNA sequencing technologies,mixture analysis,body fluid identification,phenotypic profiling,forensic genealogy,microbiological analysis,exploration of novel markers,and ethical and legal considerations.These developments have enabled the analysis of difficult samples and provided comprehensive insights into the origins of biological evidence.In an ever-evolving landscape,forensic genetics continues to shape the future of forensic science by providing new tools and techniques that help deliver justice in an increasingly complex world.
基金supported by the Queensland-Chinese Academy of Sciences(QCAS)Biotechnology Fund(GJHZ1131)the Project of Chinese Academy of Sciences for the Development of Major Scientific Research Equipment(YZ201148)+1 种基金the National Natural Science Foundation of China(31200628)the External Cooperation Program of Bureau of International Cooperation,Chinese Academy of Sciences(GJHZ201302)
文摘Research has shown that the hypomagnetic field(HMF)can affect embryo development,cell proliferation,learning and memory,and in vitro tubulin assembly.In the present study,we aimed to elucidate the molecular mechanism by which the HMF exerts its effect,by comparing the transcriptome profiles of human neuroblastoma cells exposed to either the HMF or the geomagnetic field.A total of 2464 differentially expressed genes(DEGs)were identified,216 of which were up-regulated and2248 of which were down-regulated after exposure to the HMF.These DEGs were found to be significantly clustered into several key processes,namely macromolecule localization,protein transport,RNA processing,and brain function.Seventeen DEGs were verified by real-time quantitative PCR,and the expression levels of nine of these DEGs were measured every 6 h.Most notably,MAPK1 and CRY2,showed significant up-and down-regulation,respectively,during the first 6 h of HMF exposure,which suggests involvement of the MAPK pathway and cryptochrome in the early bio-HMF response.Our results provide insights into the molecular mechanisms underlying the observed biological effects of the HMF.
基金supported by grants from the Ministerio de Economiay Competitividad of Spain(BFU2011-22825 and CSD2007-00057(TRANSPLANTA))the Generalitat Valenciana(PROMETEOII/2014/003)+2 种基金H.C.was a recipient of a Marie Curie International Reintegration Grant(PIRG03-GA-2008-231073)Research in the laboratory of H.C.was supported by a grant from the Ministerio de Economiay Competitividad of Spain(BFU2012-31719)R.C.S.held a fellowship from the Ministerio de Economfa y Competitividad of Spain(BES-2009-014106)
文摘Next-generation sequencing （NGS） technologies allow the cost-effective sequencing of whole genomes and have expanded the scope of genomics to novel applications, such as the genome-wide characterization of intraspecific polymorphisms and the rapid mapping and identification of point mutations. Next-generation sequencing platforms, such as the Illumina HiSeq2ooo platform, are now commercially available at affordable prices and routinely produce an enormous amount of sequence data, but their wide use is often hindered by a lack of knowledge on how to manipulateand process the information produced. In this review, we focus on the strategies that are available to geneticists who wish to incorporate these novel approaches into their research but who are not familiar with the necessary bioinformatic concepts and computational tools. In particular, we comprehensively summarize case studies where the use of NGS technologies has led to the identification of point mutations, a strategy that has been dubbed ＂mapping-by-sequencing＇, and review examples from plants and other model species such as Caenorhabditis elegans, Saccharomyces cerevisiae, and Drosophila mela- nogaster. As these technologies are becoming cheaper and more powerful, their use is also expanding to allow mutation identification in species with larger genomes, such as many crop plants.
基金This work was supported by the Open Research Fund from the Shanghai Key Laboratory of Forensic Medicine(Academy of Forensic Science)under Grant KF1816the Public Interest Research Grant Programs under Grant GY2020D-2.
文摘With the development and maturation of massively parallel sequencing(MPS)technology,the mitochondrial genome(mitogenome)sequencing is increasingly applied in the forensic field.In this study,we employed the strategy of short overlapping amplicons for the whole mitogenome,library preparation with tagmentation using the Nextera®XT DNA Library Preparation Kit,sequencing on the MiSeq FGxTM Forensic Genomics System and analyzing data using the mitochondrial(mtDNA)MSR Plug-in and the mtDNA Variant Analyzer.A total of 27 libraries and 56 libraries were sequenced in a run using MiSeq Reagent Kit v2 and v3,respectively.Results showed more than 1800×of averaged depth of coverage(DoC)at each position.Concordant haplotypes of 9947 A and 2800 M were obtained at 32 variants.Cross-reactivity was observed with 1 ng primate DNA and 10 ng non-primate DNA but could be easily distinguished.Full and accurate variants were obtained from at least 50 pg input DNA and from minor contributors between 19:1 and 1:19 mixed ratios with known reference profiles.More than 86%variants were detected from≥200-bp degraded samples but its haplotype was assigned to more ancestral haplogroup.Further,a total of 3962 variants were observed at 613 nucleotide positions from 103 Xibe mitogenomes with 25:1 ratio of transitions to transversions.Two new transversions(C13735A and A14755C)and two tri-alleles at nps 9824 and 16092 were identified.There were 103 unique mitogenome haplotypes from 103 Chinese Xibe that were assigned to 79 haplogroups.Haplogroup D was the preponderant top-level haplogroup in Xibe followed by F,B,M,A,N,G,C,Z,Y,HV and J.Random match probability(RMP)and haplotype diversity(HD)of the whole mitogenome was calculated as 0.0097 and 1.0000,respectively.Compared with HVS-I only,RMP decreased 33.56%,while the number of haplotypes and HD increased 15.73%and 0.49%,respectively.Principal component analysis(PCA)showed that Xibe was clustered to East and Southeast Asian.As a whole,this MPS strategy is suitable for the whole mitogenome sequencing especially for degraded samples and can facilitate generating mitogenome data to support the routine application in forensic sciences.EMP00726 is the first whole mitogenome dataset from Xibe contributed to the EMPOP.
基金supported by the Internal Security Funding Police Program of the European Commission-Directorate General MigrationHome Affairs under the European Commission[grant number HOME/2014/ISFP/AG/LAWX/4000007135].
文摘To evaluate the promising advantages of massively parallel sequencing(MPS)in our casework,we analysed a total of 33 Y-chromosomal short tandem repeats(Y-STRs)with traditional capillary electrophoresis(CE)and 25 Y-STRs using the newer MPS technology.We studied the outcome of both technologies in 64 father-son pairs using stock and custom-designed kits.Current MPS technology confirmed the 13 mutational events observed with CE and improved our understanding of the complex nature of STR mutations.By detecting isometric sequence variants between unrelated males,we show that sequencing Y-STRs using MPS can boost discrimination power.
文摘Massively parallel sequencing(MPS)offers a useful alternative to capillary electrophoresis(CE)based analysis of human identification markers in forensic genetics.By sequencing short tandem repeats(STRs)instead of determining the fragment lengths by CE,the sequence variation within the repeat region and the flanking regions may be identified.In this study,we typed 264 Uyghur individuals using the MiSeq FGx^(^(TM)) Forensic Genomics System and Primer Mix A of the ForenSeq^(^(TM)) DNA Signature Prep Kit that amplifies 27 autosomal STRs,25 Y-STRs,seven X-STRs,and 94 HID-SNPs.STRinNGS v.1.0 and GATK 3.6 were used to analyse the STR regions and HID-SNPs,respectively.Increased allelic diversity was observed for 33 STRs with the PCR-MPS assay.The largest increases were found in DYS389II and D12S391,where the numbers of sequenced alleles were 3–4 times larger than those of alleles determined by repeat length alone.A relatively large number of flanking region variants(28 SNPs and three InDels)were observed in the Uyghur population.Seventeen of the flanking region SNPs were rare,and 12 of these SNPs had no accession number in dbSNP.The combined mean match probability and typical paternity index based on 26 sequenced autosomal STRs were 3.85E36 and 1.49Eþ16,respectively.This was 10000 times lower and 1000 times higher,respectively,than the same parameters calculated from STR repeat lengths.
